This invention relates to monoclonal antibodies and their production and use.
It has been reported that some patients with rheumatoid arthritis have raised levels of antibody to the polysaccharide/peptidoglycan complex of the cell walls of Group A Streptococci (Johnson et al, Clin. exp. Immunol., 55, 115 and 61, 373). It has also been reported that mice immunised with purified rheumatoid factor, i.e. immunoglobulin, from rheumatoid arthritis patients, develop antibodies to Group A Streptococci (Johnson et al, Clin. exp. Immunol., 61, 373).
It has recently been shown that the sugar residues on immunoglobulins taken from rheumatoid arthritis patients terminate with N-acetylglucosamine (GlcNAc) significantly more frequently than do normal immunoglobulins (Parekh et al, Nature, 316, 452). It is known that N-acetylglucosamine linked to polyrhamnose is a major determinant of Group A Streptococci.
We have discovered that the N-acetylglucosamine present in the cell walls of Group A Streptococci is in essentially the same configuration as the N-acetylglucosamine present in the abnormal immunoglobulins of patients with rheumatoid arthritis. Consequently, antibodies raised against the cell walls of Group A Streptococci are capable of binding to N-acetylglucosamine residues in biological materials, e.g. the abnormal immunoglobulins of rheumatoid arthritis patients and may be used in assay methods for the detection and estimation of such biological materials.
Using this discovery, we have now produced monoclonal antibodies to the cell walls of Group A Streptococci, or more specifically to the glycoproteins having terminal N-acetylglucosamine residues present therein. Such antibodies are specific to biological materials, eg. immunoglobulins, containing terminal N-acetylglucosamine residues. Murine hybridomas capable of producing such monoclonal antibodies also form part of the invention. Such monoclonal antibodies can be used in assay methods for the purposes described below. It is particularly advantageous to be able to produce antibodies to abnormal immunoglobulins using an antigen which is not the immunoglobulin itself, since the monoclonal antibodies of the present invention can be made highly specific for abnormal immunoglobulins containing terminal N-acetylglucosamine residues and without the ability to bind other parts of immunoglobulins.
The monoclonal antibodies of the invention may be produced in accordance with generally known techniques, see for example, xe2x80x9cMonoclonal antibodies: production and maintenancexe2x80x9d by U. Lovborg (1982). William Heinemann Medical Books, London. For example, mice are immunised with respect to Group A Streptococcal cell walls. The cell wall material is injected into the mice. Spleen cells from the mice are then fused with myeloma cells, e.g. of a mouse myeloma cell line. The hybridomas thus-produced are screened for those producing antibodies possessing the correct specificity. This may be achieved by screening the antibodies by enzyme-linked immunoabsorbent assay (ELISA) on bovine serum albumin conjugated to N-acetylglucosamine (BSA-N-AG), enzyme treated fetuin (ETF) and fetuin. Fetuin is a glycoprotein found in serum which contains N-acetylglucosamine normally hidden in its structure. Treatment with sialidase and galactosidase cleaves the molecule to expose the N-acetylglucosamine and give ETF. Hybridomas producing antibodies positive both to BSA-N-AG and ETF but negative to fetuin are kept.
The novel monoclonal antibodies to N-acetylglucosamine produced in accordance with the present invention have a variety of significant utilities. They can be used more particularly to detect and assay biological materials, i.e. materials of cellular origin, usually proteins, having exposed N-acetylglucosamine residues, e.g. abnormally glycosylated immunoglobulins in body fluids taken from patients suffering from, or suspected of suffering from, rheumatoid arthritis, tuberculosis, leprosy, Crohn""s disease and similar diseases in experimental animals such as mice. The monoclonal antibodies of the invention provide an important way of detecting and estimating the presence of the abnormal immunoglobulins believed to be associated with such conditions.
The new monoclonal antibodies may also be used in the differentiation of certain types of tumour. There is evidence that tumours which bear terminal N-acetylglucosamine residues may be more susceptible to recognition by cells of the immune response, and may trigger release of cytotoxic molecules from such cells (Dennis et al, Eur. J. Biochem. (1986) 161:359-373). Receptors able to recognise GlcNAc appear to exist on myeloid cells (Ross et al., (1985), J. Immunol., 134:3307-3315; Haltiwanger and Hill, 1986, J. Biol. Chem., 261:7440-7444). A consequence of recognition of agalactosyl tumours by such cells may be a markedly reduced tendency of such tumours to metastasise. This has been demonstrated in a murine model by Dennis et al. The monoclonal antibodies of the invention can therefore be used on cancerous cells, e.g. from biopsy material, or tumours removed at surgery (e.g. in cancer of the breast), to predict the likely prognosis (i.e. their tendency to metastatize). The monoclonal antibodies of the invention do not bind to cell membranes on normal tissues, but they bind strongly to cell membranes of tumour cells having terminal N-acetylglucosamine residues on the surface, and when revealed by a suitable colour-forming label they give strong membrane staining on such cells. This has been demonstrated on a murine tumour (L929) which triggers its own destruction via cytotoxin release from macrophages which it contacts.
In clinical use, the monoclonal antibodies of the invention also make it possible to assay variations in. levels of abnormally glycosylated immunoglobulins. This assists in predicting the nature of acute abdominal crises in Crohn""s disease; assessing the efficacy of treatment in rheumatoid arthritis; monitoring the effects of immunotherapy of bladder cancer using Mycobacterium tuberculosis (var BCG); monitoring correction by immunotherapy of the immunopathological mechanisms seen in tuberculosis. The new monoclonal antibodies may also be useful in veterinary practice, for example for screening badgers for tuberculosis. As is well known, wild badgers act as a reservoir of bovine tuberculosis from which domestic cattle can be infected. To prevent indiscriminate slaughter of badgers suspected of carrying bovine tuberculosis, a simple assay method is desirable, and the monoclonal antibodies of the present invention make this possible.
For these purposes, the novel monoclonal antibodies of the present invention may be used in, for example, enzyme-linked immunoassay in the same way as known monoclonal antibodies, but relying on the specific ability of the novel antibodies to bind with N-acetylglucosamine residues generally, and especially such residues present in immunoglobulin (IgG). Enzyme-linked immunoassay is a well known technique: reference may be made, for example, to Rook and Cameron, (1981), J. Immunological Methods, 40, 109-114. By making the new monoclonal antibodies from the cell walls of Group A Streptococci rather than from the abnormal immunoglobulin of rheumatoid arthritis patients, it is possible to achieve a much higher specificity of action for N-acetylglucosamine without interference from other affinities.
In general, the monoclonal antibodies of the invention may be used to detect and/or estimate biological materials having terminal N-acetylglucosamine residues by causing the said biological material to bind to the monoclonal antibody, and to a label under conditions such that the bound or unbound label provides a measure of the said biological material, and detecting or estimating the said bound or unbound label. The label may be any of the labels currently used in immuno-assay techniques including radioactive, fluorescent and enzyme labels. Preferably an enzyme label is used along with a colour forming reagent which undergoes a chromogenic reaction catalysed by the said enzyme.
Heterogeneous assay methods are usually preferred. The immunoglobulin or other biological material to be assayed is bound to a solid support, e.g. by adsorption to an appropriate surface, the monoclonal antibody is allowed to bind to the biological material on the solid support, a label is attached directly or indirectly to said monoclonal antibody, and the said label is then assayed.
Where the biological material is a mammalian cell, e.g. from a tumour under investigation for the reasons explained above, a separate solid support is not required and the monoclonal antibody is allowed to bind to the cell itself, i.e. to any terminal N-acetylglucosamine residues present on the surface of the said cell, the label is attached directly or indirectly to any bound monoclonal antibody and the label is then observed or assayed. Single cells can be examined under the microscope in this way.
Suitable enzyme labels include peroxidase, xcex2-galactosidase, alkaline phosphatase, and glucose oxidase. In each case, a means for revealing or determining the bound enzyme label is provided in the form of a suitable substrate for the enzyme, e.g. a combination of hydrogen peroxide and a compound, e.g. o-phenylene diamine or 4-chloro-naphthol which produces a colour when oxidized by the hydrogen peroxide under the influence of the enzyme.
The label may be attached to the monoclonal antibody by an anti-mouse immunoglobulin covalently linked to the said label. Alternatively, and preferably, the monoclonal antibody of the invention is biotinylated in known manner and the label is bound thereto using avidin covalently linked to the said label.